Chondroitin sulfate (CS) is currently recommended by EULAR (the European League against Rheumatism) as a symptomatic slow-acting drug for osteoarthritis (SYSADOA) in the treatment of osteoarthritis of the knee (Jordan K M et al., Ann. Rheum. Dis. 62, 1145, 2003), hip (Jordan K M et al. Ann. Rheum. Dis. 62, 1145, 2003) and hand (Zhang W et al., Ann. Rheum. Dis. 66, 377, 2007) on the basis of numerous clinical findings and various meta-analyses of clinical trials. Recent clinical trials have also demonstrated that CS modifies the extracellular structures of human cartilage tissue (Reginster J Y, Heraud F, Zegels B, Bruyere O. Mini Rev Med Chem 7, 1051, 2007. Kahan A, Uebelhart D, De Vathaire F, Delmas P D, Reginster J Y. Arthritis Rheum 60, 524, 2009). CS is also widely used as a nutraceutical, either alone or combined with other ingredients (McAlindon T E et al., JAMA 283, 1469, 2000. Volpi N et al., Food Anal Meth 1, 195, 2008. Volpi N et al., Separation Sc 1, 22, 2009).
Chondroitin sulfate (CS) is a natural polysaccharide belonging to the glycosaminoglycan (GAG) class, present in both vertebrates and invertebrates, which consists of disaccharide sequences formed by alternating residues of glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) bonded to one another by beta 1-3 bonds and sulfated in different positions.
CS is present in animal tissues, with structural and physiological functions. It mainly consists of two types of disaccharide unit monosulfated at the 4- or 6-position of GalNAc (called disaccharides A and C respectively), present in different percentages depending on its origin. The CS backbone also contains non-sulfated disaccharide, generally in small amounts. Disulfated disaccharides having two sulfate groups bonded through the oxygen atom at various positions, such as the 2-position of GlcA and the 6-position of GalNAc (disaccharide D), the 2-position of GlcA and the 4-position of GalNac, or the 4- and 6-positions of GalNAc (disaccharide E), can be present in the CS backbone in variable percentages, depending on the specific animal sources (Volpi N. J. Pharm. Pharmacol. 61, 1271, 2009. Volpi N. J. Pharm. Sci. 96, 3168, 2007. Volpi N. Curr. Pharm. Des. 12, 639, 2006). The presence of sulfation at the 3-position of GlcA is possible, but in extremely small amounts; said presence is rare in CS of terrestrial origin, and more probable in the highly sulfated types of marine origin (Fongmoon D et al. J Biol Chem 282, 36895, 2007).
The formula of the repeating disaccharide unit of CS is as follows:

wherein R2, R4 and R6 are independently H or SO3−.
The negative charges of the carboxylate and sulfate groups in the repeating disaccharide unit are generally neutralized by sodium ions.
The meanings of the acronyms most commonly used to identify the variously sulfated disaccharides are set out below:
Di-0S (R2=H; R4=H; R6=H)
Di-6S (C) (R2=H; R4=H; R6=SO3−)
Di-4S (A) (R2=H; R4=SO3−; R6=H)
Di-4,6diS (E) (R2=H; R4=SO3−; R6=SO3−)
Di-2,6diS (D) (R2=SO3−; R4=H; R6=SO3−)
Di-2,4diS (B) (R2=SO3−; R4=SO3−; R6=H)
Di-2,4,6triS (R2=SO3−; R4=SO3−; R6=SO3−)
Samples of CS originating from different animal sources are also characterized by different molecular weights and charge densities, this latter parameter being directly correlated with the specific sulfated groups.
Table 1 shows the main disaccharides found in natural CS extracted from cartilage of various animal species.
TABLE 1CS Originating From Different Animal SourcesParametersBovine CSPorcine CSChicken CSShark CSSkate CSSquid CSMn (kDa)12-17 9-14 8-1325-4027-3460-80Mw (kDa)20-2614-2016-2150-7050-70 80-120Polydispersity1.8-2.21.4-1.81.6-2.01.0-2.01.2-2.50.8-1.3indexDi-0S 6 6 8 3 313Di-6S331420443915Di-4S618072324350Di-2,6diSNDNDND1813 0Di-4,6diSNDNDND 2 122Di-2,4diSNDNDND 1 1 0Charge0.90-0.960.92-0.960.90-0.941.15-1.251.08-1.201.00-1.20density4S/6S1.50-2.004.50-7.003.00-4.000.45-0.901.00-1.402.50-4.00ratioMn = number average molecular weight;Mw = weight average molecular weight;Polydispersity index = Mw/Mn;The charge density is the number of sulfate groups per disaccharide unit;ND = not identified
The various types of CS derived from terrestrial animals have similar molecular mass parameters (Mn and Mw), whereas they differ from those of marine species, which have higher molecular mass values. CS of terrestrial origin has a mean molecular weight (Mw) between 14 and 26 kDa, whereas CS of marine origin, obtained from squid, cartilaginous fish and bony fish, has a molecular weight (Mw) exceeding 50 kDa. Terrestrial CS samples are also characterized by charge density (CD) values below 1.0, whereas marine CS samples always have CD values exceeding 1.0.
Disulfated disaccharides are usually present in trace amounts in terrestrial CS, but are more abundant in CS of marine origin. Moreover, significant amounts of polysulfated disaccharides (tri- and tetra-sulfates) are not observed in natural CS.
Natural CS also presents differences between different organs and tissues, even in the same species, as shown in Table 2.
TABLE 2CS Derived From Different Animals and Organs/TissuesRabbit ileum,kidney, lungBovineBovineSturgeonand boneHumanHumanParameterscartilageaortabonesmarrowplateletsplasmaMn (kDa)12-17ND25-30NDNDNDMw (kDa)20-26ND35-40NDND~15 Polydispersity1.8-2.2ND1.05-1.5 NDNDNDindexDi-0S 607ND040-60Di-6S33 95-10055 ~100  Traces1-5Di-4S610-538 Traces>98 60-40Di-2,6diSND00000Di-4,6diSND00000Di-2,4diSND00000Charge density0.90-0.960.98-1.020.90-0.950.98-1.020.98-1.020.40-0.604S/6S ratio1.50-2.00 <0.10.40-0.90 <0.1>45 10-50Mn = number average molecular weight;Mw = weight average molecular weight;Polydispersity index = Mw/Mn;The charge density is the number of sulfate groups per disaccharide unit;ND = not identified.
The existence of chains of polysaccharide or oligosaccharide CS with 100% 6-sulfate or 4-sulfate disaccharides is reported in the literature for various tissues and organs (Sampaio L. O. et al. Biol. Chem. 256, 9205, 1981; Okayama E. et al. Blood 72,745, 1988; Ambrosius M. et al. J. Chrom. A 1201, 54, 2008; Volpi N. et al. Clin. Chim. Acta 370, 196, 2006).
All these characteristics demonstrate the extreme heterogeneity of natural CS in terms of both molecular weight and charge density; however, parameters according to which a CS can be defined as “natural-like” can be identified. A chondroitin 6-sulfate which has a charge density comparable to that of CS of marine origin and is characterized by the absence of abnormal sulfation patterns presents as structurally similar to natural glycosaminoglycan. Its proven anti-inflammatory activity in vivo provides further support for the definition of natural-like CS, and supports its use in the treatment of symptoms correlated with arthritic disorders.
Many attempts have been made to find a biotechnological method for the production of CS using micro-organisms as a polysaccharide precursor source having a structure partly similar to that of CS, and then using chemical sulfation to produce a CS similar to the natural type.
Some bacteria produce capsular polysaccharides with a structure similar to glycosaminoglycans; for example, Pasteurella multocida produces a polysaccharide identical to non-sulfated chondroitin (De Angelis P. L., Carbohydrate Res., 337 (17), 1547, 2002). However, the Escherichia coli strain with serotype O5:K4:H4 produces a capsular polysaccharide with a chondroitin backbone bearing a β-fructose residue bonded at the 3-position of the GlcA unit (polysaccharide K4).
An example of production of biotechnological CS starting with capsular polysaccharide K4 from E. coli O5:K4:H4 is reported in EP 1304338, which describes a process wherein polysaccharide K4, produced in liquid cultures, is extracted and purified and then redissolved and subjected to acid hydrolysis to eliminate the fructose residues bonded to the GlcA residues of the polymer. The defructosylated polymer, identical to the non-sulfated backbone of CS (CH), can be sulfated at the 4- or 6-position of the GalNAc residue according to various chemical synthesis methods, to produce a CS with a molecular weight between 6 and 25 kDa. However, the biotechnological CS described in EP 1304338 is not evaluated at all for its anti-inflammatory and anti-arthritis activity, and its use in the treatment of arthritis and/or osteoarthritis remains a mere hypothesis. This is particularly important as only 70% of the polysaccharide described in EP 1304338 definitely has the structure of a natural chondroitin sulfate, the remaining 30% being mainly non-sulfated chondroitin (CH). Furthermore, oligosaccharides with a molecular weight of less than 5 kDa are not considered.
A recent publication (Bedini E. et al. Angew Chem. Int. Ed Engl. 2011) describes a process wherein the polysaccharide K4 produced is sulfated at the 4-position and/or the 6-position of the GalNAc residue in the same chain. Once again, the biotechnological CS described is not evaluated for anti-inflammatory or anti-arthritis activity, and its use in the treatment and prevention of arthritis and/or osteoarthritis and the correlated inflammatory processes is not evaluated. The same authors postulate the presence of structural modifications to the chain of biotechnological CS deriving from their synthesis process, which produces abnormal sulfation of the hydroxyl group in C3 of GlcA due to the low protection of that group during the synthesis process. This anomaly is known to cause serious toxicity in humans following intravenous administration of heparin wherein said CS 3-sulfated in GlcA was present as a contaminant. Although this toxicity has never been observed in relation to oral administration of CS, the risk of toxic effects due to that type of anomalous sulfation remains; this is also indicated by the same authors in another recent publication (Bedini, E., et al., Chemistry: A Eur. J. (2012) vol. 18: 2123-30).
Moreover, the biotechnological CS described by Bedini et al. (Angew Chem Int Ed Engl. 2011) has a molecular weight of around 17 kDa, and therefore potentially exhibits the low bioavailability of natural products of extraction origin. For all these reasons, the biotechnological CS described by Bedini et al. is unlikely to be used in the treatment and prevention of arthritis and/or osteoarthritis.
Examples of low-molecular-weight types of CS for use in the treatment of arthritis do exist (Cho S Y et al. Biol. Pharm. Bull. 27, 47, 2004, Das A. et al. Osteoart. Cartil. 8, 343, 2000), but they are all obtained by depolymerization of CS of animal origin, which means that the presence of viruses, prions and other transmissible infectious agents cannot be ruled out. If in vivo performance of a new low-molecular-weight CS of biotechnological origin could be shown to be different than similar low-molecular-weight CS obtained from animals, particularly in the treatment of osteoarthritis, this would represent a useful contribution to the art.